Abstract Rabies disease is nearly 100% lethal in the absence of treatment, killing an estimated 59,000 people annually. When vaccines are properly administered, they are highly efficacious, making them one of the most economically high-impact interventions among infectious diseases. About 30,000 people receive post-exposure treatment in the USA annually indicating the need for treatment options against RABV. Of note, fifteen rabies-related viruses (lyssaviruses) are similarly lethal, but divergent enough to evade protection from current vaccines and biologics, which are based on the classical rabies virus (RABV). We previously designed a structurally-informed chimeric glycoprotein (G) to incorporate large ectodomain regions of two divergent lyssaviruses, RABV and Mokola virus (MOKV). In vivo, this vaccine elicited neutralizing antibodies against both RABV and MOKV and protected against challenge with viruses containing MOK G or RABV G. Based on knowledge of the RABV G, these preliminary data suggest that i) the chimeric G includes important antigenic regions from both RABV and MOKV and that ii) the elicited antibodies should protect against challenge. Moreover, some data also raised the compelling suggestion that antibody mechanisms in addition to neutralization may contribute to protection. Three independent but complementary aims are proposed to take the discoveries of the R21 to the next level. The first Aim will investigate the immunogenicity and protective quality of a chimeric G lyssavirus vaccine against a panel of different lyssaviruses of importance. Aim 2 examines antigenic regions of non-RABV lyssavirus glycoproteins (G) and delineate the antigenic regions on the lyssavirus Gs by isolating and characterizing neutralizing monoclonal antibodies (mAbs). The dogma for protection against RABV is that neutralizing antibodies are necessary and sufficient. This has not been studies for non-RABV lyssaviruses and is not completely supported by preliminary data from our previous study. Therefore, Aim 3 will revisit the mechanism of protection against Lyssaviruses in pre- and post-exposure applications and redefine the function of neutralizing and non-neutralizing antibodies for protection from rabies.